JP2001512682A - Neutral sphingomyelinase - Google Patents

Abstract

(57) Abstract The present invention relates to eukaryotic neutral sphingomyelinase (nSMase) and uses thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to eukaryotic neutral sphingomyelinase.
elinase) and its applications.

[0002] Sphingomyelin is an important component of the plasma membrane. Degradation of sphingomyelin gives some substances with potential second messenger properties such as, for example, ceramide, sphingosine or sphingosine-1-phosphate.

[0003] Bacterial neutral sphingomyelinase is a secreted, soluble protein.

The present invention provides, first of all, a nucleic acid encoding a eukaryotic neutral sphingomyelinase. Eukaryotic neutral sphingomyelinase (nSMase) is characterized in that it cleaves sphingomyelin into ceramide and phosphocholine, and its activity is dependent on the addition of magnesium ions. It is a membrane-bound enzyme.
Its maximum activity is achieved in the neutral pH range.

[0005] Preferably, the nucleic acid according to the present invention is a nucleic acid encoding a neutral sphingomyelinase of a mammal. More preferably, it encodes a neutral sphingomyelinase of human or murine. The corresponding nucleic acid sequences are disclosed in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0006] Some of the nucleic acid sequences are identical to the EST sequences AA028477 and AA013912 (murine) and W32352 and AA056024 (human).

[0007] One of skill in the art, knowing the amino acid and nucleic acid structure of human and murine neutral sphingomyelinase, will be aware of the high homology between human and murine nSMases, and will Corresponding nucleic acids and proteins can be easily found. By doing this, one of skill in the art can use cross-reactive antibodies for purification by specific affinity chromatography, or synthesize oligonucleotide primers based on nucleic acid sequences and use eukaryotes utilizing the polymerase chain reaction. of
It can either amplify the desired nucleic acid in a cDNA library. The corresponding cDNA library can be obtained in a manner known per se by isolating mRNA from a tissue sample, which is then reverse transcribed. From the nucleic acid sequence, the amino acid sequence can be obtained using the genetic code. Instead, EST (expressed sequence
tags) You can also search for homologous sequences in the database and combine them.

[0008] The nucleic acids according to the invention are suitable for the expression of eukaryotic neutral sphingomyelinase in prokaryotic or eukaryotic systems. Further, in vivo nSM in gene therapy
It is also suitable for the expression of ase, and in particular, in the form of a fragment having a complementary structure, as an antisense nucleotide for reducing the expression of nSMase.

The nucleic acids according to the invention can be produced by chemical synthesis or amplification in a genetically engineered organism by methods known per se to the person skilled in the art.

[0010] The present invention also relates to a eukaryotic neutral sphingomyelinase obtainable by expression of a nucleic acid according to the present invention.

[0011] The nSMase according to the present invention can be produced by expression in a genetically engineered organism. Eukaryotic expression systems are particularly suitable. Suitable eukaryotic expression systems are known to those skilled in the art, for example, pRc / CMV (Stratagene). Purification from genetically engineered biomass provides easy and direct access to nSMases according to the invention, especially in the case of overexpression, and also allows their isolation in large quantities.

The eukaryotic neutral sphingomyelinase is preferably a neutral sphingomyelinase of a mammal, especially a human or a murine. The amino acid sequences of human and murine neutral sphingomyelinase are represented as SEQ ID NOs: 1 and 2 (SEQ. ID. NO. 1 and 2).

The molecular weight of human and murine sphingomyelinase is 47.6 and 47.5, respectively.
kDa. Compared to the bacterial nSMase, the mammalian nSMase according to the invention does not contain a signal sequence at the N-terminus. From hydrophobicity analysis, two adjacent hydrophobic membrane regions at the C-terminus are separated by eight amino acids. Therefore, this protein is an integral membrane protein whose catalytically active region faces the cytosol while only a small portion of the enzyme is in contact with the extracellular environment.
proteins). This contrasts with the secreted soluble protein, Bacteria nSMase, but follows previous studies on the properties of neutral mammalian sphingomyelinase. According to Northern blot analysis, murine
The 1.7 kb mRNA of nSMase is expressed in all tissues. In kidney, brain, liver, heart and lung, Northern blots show strong signal, although expression in spleen appears to be low. This measurement was inconsistent with the measured enzyme activity of the corresponding cells. This indicates that there is a post-transcriptional adjustment of nSMase.

The pH optimum of the neutral sphingomyelinase according to the present invention is in the range of 6.5 to 7.5 when the Km value of C18 sphingomyelin is in the range of 1.0 to 1.5 × 10 ⁻⁵ M. This activity depends on the presence of magnesium ions. Addition of EDTA results in suppression of the activity of SMase, but can be restored by adding Mn ²⁺ or Mg ²⁺ ions. Addition of 0.3-0.5% Triton X-100 increases enzyme activity. This activity is unaffected by treatment with DTT or 2-mercaptoethanol, whereas it is inhibited by the addition of 20 mM glutathione. n
Although the activity of SMase is not restricted to sphingomyelin, the structurally related phosphatidylcholine is also cleaved with approximately 3% activity.

[0015] Variants of eukaryotic neutral sphingomyelinase are also claimed. The term "variant" is used to describe naturally occurring allelic changes in eukaryotic neutral sphingomyelinase and production by recombinant DNA technology, particularly by in vitro mutagenesis using chemically synthesized oligonucleotides. And proteins in which expression corresponding to eukaryotic neutral sphingomyelinase with respect to biological and / or immunological activity has occurred. This may include amino acid deletions, insertions or conservative substitutions. "Conservative substitution" means that an amino acid is replaced by another amino acid having similar physicochemical properties.

Thus, for example, the following amino acids can be exchanged: Serine and alanine,
Alanine and glycine, methionine and serine, lysine and arginine, lysine and serine.

In particular, the term “variant” includes acetylated, glycosylated, amidated and / or phosphorylated derivatives as well as N-terminal and / or C-terminal truncated proteins.

At least part of the activity of nSMase is that fragment 1-282 of murine nSMase
It did not show an increase in sphingomyelinase activity when expressed in EK293 cells, and therefore appears to belong to the C-terminal region. The present invention also provides nSMas
For the C-terminal fragment of e. Compounds in which nSMase or a variant thereof is conjugated to another molecule such as, for example, a dye, a radionuclide or an affinity component are also variants according to the invention.

Nucleic acids encoding eukaryotic neutral sphingomyelinase or complementary to such nucleic acids are also claimed. The nucleic acid may be, for example, DNA, RNA, PNA or a nuclease resistant analog thereof. In particular, nuclease-resistant analogs include compounds having a phosphodiester bridge modified by a hydrolytically stable compound, such as phosphothioate, methylphosphonate, and the like.

In particular, short fragments of the nucleic acids are suitable as antisense nucleotides. For reasons of specificity, they should preferably contain 6 or more nucleotides, more preferably 8 or more, most preferably 12 or more nucleotides. For reasons of prevalence and cost, they usually have a length of less than 30 nucleotides, preferably 24 or less, more preferably 18 or less.

The present invention relates to derivatives of nucleic acids, fragments of nucleic acids according to the invention, complementary to these nucleic acids, which are linked to other molecules for diagnostic or therapeutic purposes, such as fluorescent dyes, radioactive labels or affinity components. Specific nucleic acids and variants of said nucleic acids.

As used herein, “fragment” refers to a nucleic acid that has been truncated at the 5 ′ or 3 ′ or both ends. The term "variant" means that these nucleic acids hybridize with the nucleic acids according to the invention or nucleic acids complementary thereto under stringent conditions.
The term “harsh environment” means that hybridization is carried out under conditions where the temperature is 10 ° C. lower than a temperature just low enough to anneal exactly complementary nucleic acids (other conditions being identical). Means to be For example, if an exactly complementary nucleic acid is annealed down to about 55 ° C. under given conditions, then the harsh conditions are temperatures above 45 ° C. Preferably, the stringent temperature range is within 5 ° C, more preferably within 3 ° C.

Furthermore, the present invention relates to an antibody against the nSMase according to the present invention or the nucleic acid according to the present invention. These substances are particularly suitable as medicaments for diagnosis or for use in immunoassays known per se to the person skilled in the art, for histology and for treating conditions associated with overexpression of nSMase. Such antibodies according to the invention are obtained by vaccination with the nSMases or nucleic acids or peptides and nucleic acid fragments according to the invention in the presence of an adjuvant by methods known per se to the person skilled in the art.

The invention furthermore relates to a cell line overexpressing the nSMase according to the invention. Such a cell line is obtained by transfer of a vector comprising a nucleic acid according to the invention encoding nSMase. In the case of eukaryotic cell lines, for example, the transfer may be effected by electroporation. Preferably, the cell line is stably transferred.

In this context, “overexpression” means that the cell line has a higher activity of nSMase than the cell line into which the nucleic acid according to the invention has not been transferred. For example, suitable eukaryotic cell lines include the cell lines U937, HEK293 or Jurkat.

In experiments, cell lines showed specific nSMase activity between 0.3 and 10 μmol / mg of protein per unit time.

FIG. 10 shows Northern and Western blot analysis of nSMase expression in transfected cell lines. Site A shows the results of RT PCR of total cellular RNA using primers that hybridize with human and murine nSMase cDNA. Site B shows T PCR of total RNA using a primer that hybridizes with human β-actin cDNA as a control. Site C shows a western blot of plasma membrane cell extracts of different HEK293 cell lines after SDS polyacrylamide gel electrophoresis and hybridization with a polyclonal anti-nSMase antibody.

Addition of 0.5 mM arachidonic acid resulted in a 3-fold increase in nSMase activity in HEK cell overexpression.

The present invention further relates to a transgenic mammal exhibiting overexpression (gain of function) or genetic deficiency or deficiency (loss of function) for the nSMase according to the invention. The mammal is preferably a rodent, especially a mouse. Such transgenic mammals can be obtained by methods known per se to those skilled in the art and are particularly suitable for elucidating the function of neutral sphingomyelinase. For transgenic mammals, a given gene construct is injected into the pronucleus of a fertilized egg by DNA microinjection to achieve expression of the additional gene. Selective changes in genes in the genome of ES cells that are subsequently injected into the blastocyst
Switch off the function of the gene.

The strategy and construction for the generation of mouse mutants is shown in FIGS.

The transgenic animal is preferably an animal capable of switching genes on and off temporarily in a tissue-specific manner by external induction. Such transgenic animals are particularly suitable for elucidating the metabolic and single transduction pathway for nSMase according to the invention. This in turn allows for diagnostic or therapeutic applications. In particular, transgenic mammals are suitable for screening for pharmaceutically active substances.

The eukaryotic neutral sphingomyelinase according to the invention, the nucleic acid according to the invention and the antibody according to the invention are contained in medicaments and diagnostics, optionally further with auxiliaries. Such medicaments and diagnostics may be used for diseases based on over- or under-expression and / or increased or decreased activity of eukaryotic intermediate sphingomyelinase and / or disorders of cell proliferation, differentiation, and / or apoptosis. Suitable for diagnosis and treatment.

In particular, these are diseases involving inflammatory processes, cell growth disorders, and metabolic disorders. For example, they can be cancer or cholesterol homeostasis (atherosclerosis).

The pharmaceutical screening method according to the invention relies on a change in the expression or activity of an nSMase according to the invention in an nSMase-overexpressing cell line, which coincides with the addition of at least one potential pharmaceutically active substance. Therefore, the cell line is particularly suitable for the development and testing of drug-derived structures.

The present invention is further illustrated in the following examples.

【Example】

Example 1 Cloning of Nucleic Acid A nucleic acid of the present invention encoding neutral sphingomyelinase was cloned into a cloning site and a NotI restriction site of a eukaryotic expression vector pRc / CMV (Stratagene). The sequence of the obtained DNA was determined by a sequencing method using a Perkin Elmer DNA sequencer 377A.

Example 2 Cloning of RNA Total RNA was isolated by known methods from different organs of eight 3-week-old CD1 rats, and poly (A ⁺ ) RNA was isolated from oligo (dT) cellulose (Boehringer Mannheim). (Boehringer Man
nheim), Germany) and isolated by standard methods with affinity purification.

Example 3 U937 cells were cultured in PRMI 1640 medium containing 10% fetal bovine serum, 1 μg / ml penicillin / streptomycin and 0.03% glutamine at 37 ° C. under 5% CO ₂ . By electroporation using a gene pulsar (Bio-Rad), 5 × 10 ⁶ cells were transfected together with 1 μg of linear plasmid DNA encoding the nSMase of the present invention. Selection of stable clones was performed using 1 mg / ml geneticin (G418,
Life Technologies, Gaithersburg, MD).

[0038] The nSMase purified from the cell line had a specific activity of between 0.3 and 10 μmol / mg protein / hour. The optimum pH values were 6.5 and 7.5. K _M values of C18 sphingomyelin phosphorus was 1.0 to 1.5 × 10 ^-5 M. The activity was dependent on the presence of magnesium ions, and the activity was inhibited by the addition of EDTA.

Example 4 Measurement of nSMase Activity Enzymatic activity was tested in cells and murine tissues. Cells were washed twice with ice-cold PBS and precipitated at 1000 × g. The pellet was resuspended in cell lysis buffer and cells were broken by repeated freeze and thaw cycles. After centrifugation at 2500 × g for 2 minutes, extraction with cell lysis buffer containing 0.2% Triton X-100 was performed, followed by centrifugation at 100,000 × g for 15 minutes.

[0040] Three week old murine tissues were homogenized in cold cell lysis buffer. The tested amounts of protein and homogenized tissue were brought to a final volume of 200 μl with 10 nM (80,000 dpm) [N- ¹⁴ CH ₃ ] sphingomyelin and incubated for 30 minutes at 37 ° C. Thereafter, 100 μl of water was added and the unreacted substrate was removed by chloroform-methanol (2: 1, v / v) extraction. The radioactivity of the aqueous phase containing the enzymatically released phosphocholine was measured in a scintillation counter.

Example 5 Polyclonal Antibodies Rabbits were immunized with the synthetic peptide CDPHSDKPFSDHE (corresponding to amino acids 261 to 273 of murine nSMase) coupled to keyhole limpet hemocyanin. The polyclonal antibody serum was purified by chromatography using hydroxyapatite and affinity chromatography using a column to which the above-mentioned synthetic peptide binds.

[Sequence list]

[Brief description of the drawings]

FIG. 1. Gene sequence of human neutral sphingomyelinase.

FIG. 2: Gene sequence of human neutral sphingomyelinase.

FIG. 3. Gene sequence of human neutral sphingomyelinase.

FIG. 4: Gene sequence of murine neutral sphingomyelinase.

FIG. 5 shows the gene sequence of murine neutral sphingomyelinase.

FIG. 6 shows the gene sequence of murine neutral sphingomyelinase.

FIG. 7 shows the gene sequence of murine neutral sphingomyelinase.

FIG. 8. Gene sequence of murine neutral sphingomyelinase.

FIG. 9 shows the gene sequence of murine neutral sphingomyelinase.

FIG. 10 shows the results of Northern and Western blots of an nSMase overexpressing cell line.

FIG. 11. Strategy for producing murine knockout mutants. Letters indicate restriction sites.

FIG. 12. Design for obtaining transgenic mouse mutants.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] February 14, 2000 (2000.2.14)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 9/10 A61P 35/00 4C085 29/00 C07K 16/40 4H045 35/00 C12N 1/15 C07K 16 / 40 1/19 C12N 1/15 1/21 1/19 9/16 D 1/21 G01N 33/50 T 5/10 C12N 15/00 ZNAA 9/16 A61K 37/54 G01N 33/50 C12N 5/00 B A (31) Priority claim number 60 / 078,386 (32) Priority date March 18, 1998 (March 18, 1998) (33) Priority claim country United States (US) (81) Designated country EP ( AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, M , MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AU, BA, BB, BG, BR, CA, CN, CU, CZ, DE, EE, GE, HR, HU, ID, IL, IS, JP, KP, KR, LC, (72) Invention of LK, LR, LT, LV, MG, MK, MN, MX, NO, NZ, PL, RO, SG, SI, SK, SL, TR, TT, UA, US, UZ, VN, YU Hoffman Kai Germany Federal Republic of Cologne D-50931, Josef-Stertsmann-Strase 52, Med Fak, Institute Fuer Biochemy, Labratorium Fuhr Molecular Neurowissen Shakhten (72) Inventor Tommi Stefand Cologne D-50931, Josef-Steltzmann-Strase 52, Med Fak, Institute of Biochemistry, Labratrium Fure Molecular Neurovissen-Schaften F-term (Reference) 2G045 DA12 DA13 FB02 FB03 4B024 AA01 CA04 CA12 DA02 GA14 HA01 4B050 CC03 DD11 LL01 LL03 4B065 AA91Y AA94X AC14 BA02 CA31 CA44 CA46 4C084 AA01 AA02 AA06 AA13 BA44 CA53 CA59 DA39 DC22 ZA452 ZB112 ZB212 ZB262 ZC332 4C085 AA11 BB22A33 DD33

Claims (24)

[Claims] 1. A nucleic acid encoding a eukaryotic neutral sphingomyelinase. 2. The nucleic acid according to claim 1, wherein the nucleic acid encodes a neutral sphingomyelinase of a mammal, particularly a neutral sphingomyelinase of a human or mouse. 3. The method according to claim 1, wherein said neutral sphingomyelinase has an ability to cleave sphingomyelin into ceramide and phosphocholine, and its activity is dependent on the addition of magnesium ions. The nucleic acid according to Item. (4) SEQ ID NO: 3 (SEQ. ID. NO. 3) or SEQ ID NO.
ID. NO. The nucleic acid according to at least one of claims 1 to 3, which has the sequence according to (4). 5. The nucleic acid according to claim 1, which is DNA, RNA, PNA or a nuclease-resistant analog thereof. 6. The nucleic acid according to claim 1, which is an mRNA, a cDNA or a genomic DNA. 7. A nucleic acid which is complementary to the nucleic acid according to at least one of claims 1 to 5. 8. The nucleic acid according to claim 1, especially SEQ ID NO: 1 (SEQ. ID. NO.
. 1) or a eukaryotic neutral sphingomyelinase obtainable by expression of a nucleic acid having the sequence described in SEQ ID NO: 2 (SEQ. ID. NO. 2). 9. The eukaryotic neutral sphingomyelinase according to claim 8 or the eukaryotic neutral sphingomyelinase according to claim 8.
An antibody directed against the nucleic acid of at least one of claim 7. 10. A cell line overexpressing the eukaryotic neutral sphingomyelinase according to claim 8. 11. The cell line U937, which expresses eukaryotic neutral sphingomyelinase and
11. The cell line according to claim 10, which is a cell line based on HEK293 or Jurkat. 12. A transgenic mammal showing overexpression (gain of function) or genetic deficiency or deficiency (loss of function) for eukaryotic neutral sphingomyelinase. 13. The transgenic mammal according to claim 12, which is a rodent. 14. An eukaryotic neutral sphingomyelinase according to claim 8, a nucleic acid according to at least one of claims 1 to 7, and / or an antibody according to claim 9 together with a further auxiliary agent. Medicine. 15. An eukaryotic neutral sphingomyelinase according to claim 8, a nucleic acid according to at least one of claims 1 to 7, and / or an antibody according to claim 9 together with further auxiliary agents. Diagnostics to do. 16. Diagnosis and treatment of diseases based on over- or under-expression and / or increase or decrease of eukaryotic neutral sphingomyelinase activity and / or disorders of cell proliferation, cell differentiation and / or apoptosis. Use of the medicine according to claim 14 or the diagnostic agent according to claim 15. 17. The method according to claim 15, wherein the disease is an inflammatory process, a cell growth disorder, cancer and / or a metabolic disorder such as cholesterol homeostasis (atherosclerosis).
Use as described in. 18. The method according to claim 10, wherein the addition of at least one potentially pharmaceutically active substance is simultaneous.
A method for screening for an active substance, which comprises measuring a change in expression or activity of eukaryotic neutral sphingomyelinase in the cell line described in (1). 19. Use of the cell line according to claim 10 for developing and testing drug-derived structures. 20. The method for preparing a eukaryotic neutral sphingomyelinase according to claim 8, wherein the eukaryotic neutral sphingomyelinase is prepared by chemical peptide synthesis or by expression in a genetically designed organism, particularly a eukaryotic expression system. 21. The method for preparing a nucleic acid according to at least one of claims 1 to 7, by chemical synthesis or by amplification in a genetically engineered organism. 22. A coding region (exon), particularly SEQ ID NO: 5 (SEQ. ID. NO.
. 5) or a eukaryotic neutral sphingomyelinase gene containing a non-coding region (intron) in addition to the gene having the sequence described in SEQ ID NO: 6 (SEQ. ID. NO. 6). Item 6. The nucleic acid according to Item 5. 23. A mutant of the neutral sphingomyelinase according to claim 8. 24. The nucleic acid according to claim 1, which is a derivative, a fragment or a mutant of such a nucleic acid.